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Hybridoma cell strain capable of secreting bisamides resistant monoclonal antibody and application thereof

A hybridoma cell line, monoclonal antibody technology, applied in the field of immunochemistry, can solve the problems of expensive equipment, high solvent, consumption and so on

Active Publication Date: 2021-12-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the high sensitivity and specificity of these chromatography-based methods, there are some disadvantages such as the need for thorough sample cleanup, high solvent consumption, expensive equipment, and skilled technicians

Method used

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  • Hybridoma cell strain capable of secreting bisamides resistant monoclonal antibody and application thereof
  • Hybridoma cell strain capable of secreting bisamides resistant monoclonal antibody and application thereof
  • Hybridoma cell strain capable of secreting bisamides resistant monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Synthesis of Hapten (Hapten 1)

[0061] Since small molecules are not immunogenic and cannot stimulate mice to produce an immune response to produce antibodies, it is necessary to couple small molecules to proteins through protein linkage technology to obtain immunogenicity; commonly used in protein coupling techniques Active groups include amino, carboxyl, hydroxyl, mercapto, etc. In order to obtain monoclonal antibodies that can specifically recognize cyclobromide, cyantraniliprole, chlorantraniliprole, and tetrachlorantraniliprole, it is necessary to design and derive a For a good hapten, the specific derivatization steps are as follows.

[0062] Step 1: Synthesis of tert-butyl 4-(2-amino-3-bromo-5-chlorobenzamido)butyrate

[0063]

[0064] Add 1.0g (4.0mmol) 2-amino-3-bromo-5-chlorobenzoic acid, 10ml toluene and 0.80mL (12.0mmol) thionyl chloride to the reaction flask, reflux for 3 hours, remove the solvent, add 5mL tetrahydrofuran and wait Use; the ...

Embodiment 2

[0068] Example 2: Synthesis of Complete Antigen

[0069] Weigh 2.8mg hapten (Hapten 1), 1.6mg N-hydroxysuccinimide (NHS), dissolve in 300μL N,N-dimethylformamide (DMF), stir at room temperature for 10min; then weigh 2.6mg 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolved with 100μL DMF, added to the hapten solution, stirred at room temperature for 6-8h (called Liquid A). Take 10mg of BSA, dilute it to 5mg / mL with 0.01M carbonate buffer (CBS) (referred to as solution B), then slowly add solution A to solution B drop by drop, react at room temperature overnight; then use 0.01M PBS solution Dialyzed to remove unreacted small molecular hapten to obtain the complete antigen, which was identified by UV absorption scanning method.

Embodiment 3

[0070] Embodiment 3: the synthesis of coating original

[0071]In order to improve the sensitivity, a new coating hapten (Hapten2) structure is designed as follows:

[0072]

[0073] The synthesis method of hapten (Hapten2) is similar to that of hapten (Hapten1). The specific steps for the synthesis of the coating agent are as follows: dissolve 3.6 mg hapten (Hapten2) and 2.3 mg N-hydroxysuccinimide (NHS) in 300 μL of anhydrous N,N-dimethylformamide (DMF), and stir the reaction at room temperature After 10 min, the hapten solution was obtained; 3.8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL of anhydrous DMF, and added to the hapten solution , stirred at room temperature and reacted for 6-8 hours to obtain liquid A; dilute 10 mg of chicken ovalbumin (OVA) with 1 mL of carbonate buffer solution (CBS) with a concentration of 0.01mol / L to obtain liquid B; Slowly add it into liquid B for reaction to obtain a reaction liquid;...

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Abstract

The invention provides a hybridoma cell strain capable of secreting a bisamides resistant monoclonal antibody and an application thereof, and belongs to the technical field of immunochemistry. The preservation number of the hybridoma cell strain is CGMCC No.22322. The preparation method comprises the following steps: mixing and emulsifying a complete antigen and a Freund's adjuvant, carrying out subcutaneous immunization on a mouse, fusing splenocytes from the high-titer and low-IC50 mouse with myeloma cells of the mouse through a PEG method, and screening out hybrid cells after fusing the two cells by adopting a selective culture medium; and screening cells by an indirect competitive enzyme-linked immunosorbent assay and subcloning for three times to finally obtain the hybridoma cell strain secreting the monoclonal antibody The monoclonal antibody secreted by the cell strain has good detection sensitivity to cyclaniliprole, cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole, and can be used for detecting residues of the four pesticides in foods.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, in particular to a hybridoma cell strain secreting anti-bisamide compound monoclonal antibody and application thereof. Background technique [0002] Cyclaniliprole, cyantraniliprole, chlorantraniliprole, and tetrachlorantraniliprole are all bisamide insecticides. Bisamide insecticides are ryanodine receptor activators, which have excellent control effects on lepidopteran pests on rice and other crops, are safe to non-target organisms, and are incompatible with other existing insecticides with other modes of action. cross resistance. At present, 4 varieties are commercialized, namely flubendiamide, which was launched in 2006, chlorantraniliprole, which was launched in 2007, cyantraniliprole, which was launched in 2013, and tetrachlorantraniliprole, which was launched in 2014. [0003] Chlorantraniliprole was discovered by DuPont with flubendiamide as the lead compound after in-depth pilo...

Claims

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Application Information

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IPC IPC(8): C12N5/20C12N15/06C07K16/44G01N33/577G01N33/53
CPCC07K16/44C12N5/163G01N33/577G01N33/5308C07K2317/33G01N2430/10
Inventor 胥传来郭鑫匡华刘丽强宋珊珊胡拥明吴爱红徐丽广郝昌龙孙茂忠马伟
Owner JIANGNAN UNIV
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